Phenol oxidizing enzymes

ABSTRACT

Disclosed herein are novel phenol oxidizing enzymes naturally-produced by strains of the species  Stachybotrys  which possess a pH optima in the alkaline range and which are useful in modifying the color associated with dyes and colored compounds, as well as in anti-dye transfer applications. Also disclosed herein are biologically-pure cultures of strains of the genus Stachybotrys, designated herein  Stachybotrys parvispora  MUCL 38996 and  Stachybotrys chartarum  MUCL 38898, which are capable of naturally-producing the novel phenol oxidizing enzymes. 
     Disclosed herein is the amino acid and nucleic acid sequence for  Stachybotrys  phenol oxidizing enzyme B as well as expression vectors and host cells comprising the nucleic acid. Disclosed herein are methods for producing the phenol oxidizing enzyme as well as methods for constructing expression hosts.

FIELD OF THE INVENTION

The present invention relates to novel phenol oxidizing enzymes, inparticular, novel phenol oxidizing enzymes derived from strains ofStachybotrys and novel strains of the genus Stachybotrys producing theseenzymes. The present invention provides methods and host cells forexpressing Stachybotrys phenol oxidizing enzymes as well as methods forproducing expression systems.

BACKGROUND OF THE INVENTION

Phenol oxidizing enzymes function by catalyzing redox reactions, i.e.,the transfer of electrons from an electron donor (usually a phenoliccompound) to molecular oxygen (which acts as an electron acceptor) whichis reduced to H₂O. While being capable of using a wide variety ofdifferent phenolic compounds as electron donors, phenol oxidizingenzymes are very specific for molecular oxygen as the electron acceptor.

Phenol oxidizing enzymes can be utilized for a wide variety ofapplications, including the detergent industry, the paper and pulpindustry, the textile industry and the food industry. In the detergentindustry, phenol oxidizing enzymes have been used for preventing thetransfer of dyes in solution from one textile to another duringdetergent washing, an application commonly referred to as dye transferinhibition.

Most phenol oxidizing enzymes exhibit pH optima in the acidic pH rangewhile being inactive in neutral or alkaline pHs.

Phenol oxidizing enzymes are known to be produced by a wide variety offungi, including species of the genii Aspergillus, Neurospora,Podospora, Botytis, Pleurotus, Fomes, Phlebia, Trametes, Polyporus,Rhizoctonia and Lentinus. However, there remains a need to identify andisolate phenol oxidizing enzymes, and organisms capable ofnaturally-producing phenol oxidizing enzymes, which present pH optima inthe alkaline range for use in detergent washing methods andcompositions.

SUMMARY OF THE INVENTION

The present invention relates to novel phenol oxidizing enzymes. In apreferred embodiment, the present invention relates to phenol oxidizingenzymes obtainable from Stachybotrys. In particular, the enzymes of thepresent invention are capable of modifying the color associated withdyes and colored compounds having different chemical structures,especially at neutral or alkaline pH. Based on their color modifyingability, phenol oxidizing enzymes of the present invention can be used,for example, for pulp and paper bleaching, for bleaching the color ofstains on fabric and in detergent and textile applications. In oneaspect of the present invention, the phenol oxidizing enzyme is able tomodify the color of a dye or colored compound in the absence of anenhancer. In another aspect of the present invention, the phenoloxidizing enzyme is able to modify the color in the presence of anenhancer.

The present invention is based upon the identification andcharacterization of a genomic sequence (SEQ ID NO:3) encoding a phenoloxidizing enzyme obtainable from Stachybotrys and having the deducedamino acid sequence as shown in SEQ ID NO:2.

Accordingly, the present invention provides phenol oxidizing enzymescomprising between at least 68% and 100% identity, that is, at least 68%identity, at least 70%, at least 75% identity, at least 80% identity, atleast 85% identity, at least 90% identity and at least 95% identity tothe phenol oxidizing enzyme having the amino acid sequence disclosed inSEQ ID NO:2, as long as the enzyme is capable of modifying the colorassociated with dyes or colored compounds. In one embodiment, the phenoloxidizing enzyme has the amino acid sequence as shown in SEQ ID NO:2 oras contained in Stachybotrys chartarum having MUCL accession number38898.

In one embodiment, the phenol oxidizing enzyme is obtainable from aStachybotrys species including Stachybotrys parvispora, Stachybotryschartarum; S. kampalensis; S. theobromae; S. bisbyi, S. cylindrospora,S. dichroa, S. oenanthes and S. nilagerica. In another embodiment, theStachybotrys includes Stachybotrys chartarum MUCL 38898 and S. chartarumMUCL 30782.

In yet another embodiment, the present invention provides an isolatedpolynucleotide encoding a phenol oxidizing enzyme wherein saidpolynucleotide comprises a nucleic acid sequence having between at least65% and 100% identity, that is, at least 65% identity, at least 70%, atleast 75% identity, at least 80%, at least 85%, at least 90% and atleast 95% identity to SEQ ID NO:1, as long as the polynucleotide encodesa phenol oxidizing enzyme capable of modifying the color associated withdyes or colored compounds. The present invention encompassespolynucleotide sequences that hybridize under conditions of highstringency to the polynucleotide shown in SEQ ID NO:1 or SEQ ID NO:3 aslong as the sequence is capable of modifying the color associated withdyes or colored compounds. The present invention also encompassespolynucleotides that encode the amino acid sequence as shown in SEQ IDNO:2. In one embodiment, the polynucleotide has the nucleic acidsequence as shown in SEQ ID NO: 1 or SEQ ID NO:3 or as contained inStachybotrys chartarum having MUCL accession number 38898. The presentinvention also provides expression vectors and host cells comprisingpolynucleotides of the present invention.

The present invention additionally relates to methods for producing aphenol oxidizing enzyme of the present invention. Accordingly, thepresent invention provides a method for producing a phenol oxidizingenzyme comprising the step of culturing a host cell comprising anisolated polynucleotide encoding a phenol oxidizing enzyme having asequence comprising between at least 68% and 100% identity, that is, atleast 68% identity, at least 70%, at least 75% identity, at least 80%identity, at least 85% identity, at least 90% identity and at least 95%identity to the phenol oxidizing enzyme having the amino acid sequencedisclosed in SEQ ID NO:2 under conditions suitable for the production ofsaid phenol oxidizing enzyme; and optionally recovering said phenoloxidizing enzyme produced. In one embodiment, the polynucleotidecomprises the sequence as shown in SEQ ID NO:1. In another embodiment,the polynucleotide comprises the sequence as shown in SEQ ID NO: 3. Inan additional embodiment, the polynucleotide hybridizes under conditionsof high stringency with the polynucleotide having the sequence as shownin SEQ ID NO:1 or SEQ ID NO:3 or as contained in Stachybotrys chartarumhaving MUCL accession number 38898. In a further embodiment, thepolynucleotide has between 65% and 100%, that is, at least 65% identity,at least 70%, at least 75% identity, at least 80% identity, at least 85%identity, at least 90% identity and at least 95% identity to SEQ ID NO:1 or SEQ ID NO:3.

The present invention also provides a method for producing a recombinanthost cell comprising a polynucleotide encoding a phenol oxidizingenzyme, comprising the steps of obtaining an isolated polynucleotideencoding said phenol oxidizing enzyme said polynucleotide having betweenat least 65% and 100% identity, that is, at least 65% identity, at least70%, at least 75% identity, at least 80%, at least 85%, at least 90% andat least 95% identity to SEQ ID NO:3; introducing said polynucleotideinto said host cell; and growing said host cell under conditionssuitable for the production of said phenol oxidizing enzyme. In oneembodiment, the polynucleotide is integrated into the host genome and inanother embodiment, the polynucleotide is present on a replicatingplasmid. The present invention also encompasses polynucleotide sequencesthat hybridize under conditions of high stringency to the polynucleotideshown in SEQ ID NO:1 or SEQ ID NO:3. The present invention also providespolynucleotides that encode the amino acid sequence as shown in SEQ IDNO:2. In one embodiment, the polynucleotide has the nucleic acidsequence as shown in SEQ ID NO:1 or SEQ ID NO:3 or as contained inStachybotrys chartarum having MUCL accession number 38898.

In one embodiment of the present invention, the host cell comprising apolynucleotide encoding a phenol oxidizing enzyme includes filamentousfungus, yeast and bacteria. In another embodiment, the host cell is afilamentous fungus including Aspergillus species , Trichoderma speciesand Mucor species. In an additional embodiment, the filamentous fungushost cell includes Aspergillus niger var. awamori and Trichodermareseei.

In another embodiment of the present invention, the host cell is a yeastwhich includes Saccharomyces, Pichia, Hansenula, Schizosaccharomyces,Kluyveromyces and Yarrowia species. In yet another embodiment, theSaccharomyces species is Saccharomyces cerevisiae. In an additionalembodiment, the host cell is a bacteria including gram positivebacteria, such as a Bacillus species, and gram negative bacteria, suchas an Escherichia species.

Also provided herein are enzymatic compositions comprising the aminoacid having between at least 68% and 100% identity, that is, at least68% identity, at least 70%, at least 75% identity, at least 80%identity, at least 85% identity, at least 90% identity and at least 95%identity to the phenol oxidizing enzyme having the amino acid sequencedisclosed in SEQ ID NO:2. In one embodiment, the amino acid has thesequence as shown in SEQ ID NO: 2. Such enzymatic compositions can beused, for example, for producing detergents and other cleaningcompositions; compositions for use in pulp and paper applications; andtextile applications.

The present invention also encompasses methods for modifying the colorassociated with dyes or colored compounds which occur in stains onsamples, comprising the steps of contacting the sample with acomposition comprising an amino acid having a sequence between at least68% and 100% identity, that is, at least 68% identity, at least 70%, atleast 75% identity, at least 80% identity, at least 85% identity, atleast 90% identity and at least 95% identity to the phenol oxidizingenzyme having the amino acid sequence disclosed in SEQ ID NO:2, as longas the enzyme is capable of modifying the color associated with dyes orcolored compounds. In a preferred embodiment of the method, the aminoacid is that shown in SEQ ID NO:2.

In one aspect of the method, the pH optimum is between 5.0 and 11.0, inanother aspect, the pH optimum is between 7 and 10.5 and in yet anotheraspect the pH optimum is between 8.0 and 10. In a further aspect of themethod, the optimum temperature is between 20 and 60 degrees C. and inanother aspect between 20 and 40 degrees C.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides the nucleic acid sequence (SEQ ID NO:1) for a phenoloxidizing enzyme obtainable from Stachybotrys chartarum by PCR asdescribed in Example 5.

FIG. 2 provides the amino acid sequence (SEQ ID NO:2) for the amino aciddesignated herein as the Stachybotrys oxidase B gene.

FIG. 3 illustrates the genomic sequence (SEQ ID NO:3) for a phenoloxidizing enzyme obtainable from Stachybotrys chartarum. This nucleicacid sequence is referred to herein as Stachybotrys oxidase B gene.

FIG. 4 is an amino acid alignment of Stachybotrys phenol oxidase Benzyme SEQ ID NO:2 (bottom line) and Bilirubin oxidase (SEQ ID NO:4).

FIG. 5 provides an illustration of the vector pGAPT2-spoB which was usedfor the expression of Stachybotrys phenol oxidizing enzyme inAspergillus. Base 1 to 1134 contains Aspergillus niger glucoamylase genepromoter. Base 3098 to 3356 and 4950 to 4971 contains Aspergillus nigerglucoamylase terminator. Aspergillus nidulans pyrG gene was insertedfrom 3357 to 4949 as a marker for fungal transformation. The rest of theplasmid contains pBR322 sequences for propagation in E. coli. Nucleicacid encoding the Stachybotrys phenol oxidizing enzyme of SEQ ID NO:1was cloned into the Bgl II and Age I restriction sites.

FIG. 6 is an illustration of expression of the Stachybotrys oxidase Bprotein in a replicating plasmid. The Stachybotrys oxidase expression isunder the Aspergillus glucoamylase promoter and terminator control. Thetransformation marker pyrG gene and the AMA 1 sequence are fromAspergillus nidulans.

FIG. 7 shows the pH profile for Stachybotrys oxidase B against thesubstrate 2,6 DMP.

FIG. 8 shows a non-denatured (native) gel electrophoresis ofStachybotrys chartarum fractions from an ion exchange column (silverstained) as described in Example 1.

FIG. 9 shows a non-denatured gel electrophoresis of fractions with ABTSoverlay as described in Example 1.

FIG. 10 shows an SDS-PAGE gel of bands identified and isolated from anABTS overlay of the gel shown in FIG. 9. Stachybotrys chartarum oxidaseB is shown in the lane labeled Overlay 2. Lane Overlay 2 shows theobserved banding pattern for Stachybotrys chartarum oxidase B under theconditions described.

DETAILED DESCRIPTION Definitions

As used herein, the term phenol oxidizing enzyme refers to those enzymeswhich are capable of catalyzing redox reactions wherein the electrondonor is a phenolic compound and which are specific for molecular oxygenor hydrogen peroxide as the electron acceptor. One illustrative phenoloxidizing enzyme of the present invention obtainable from Stachybotryschartarum is shown in SEQ ID NO:2. The present invention encompassesphenol oxidizing enzymes that have between at least 68% and 100%identity, that is, at least 68% identity, at least 70%, at least 75%identity, at least 80% identity, at least 85% identity, at least 90%identity and at least 95% identity to the phenol oxidizing enzyme havingthe amino acid sequence disclosed in SEQ ID NO:2. As used herein,identity is measured by the GAP program of GCG software (UniversityResearch Park, Madison Wis.) with the following parameters: GapWeight=12; Length Weight=4; Gap Creation Penalty=8; and Gap ExtensionPenalty=2.

As used herein, Stachybotrys refers to any Stachybotrys species whichproduces a phenol oxidizing enzyme capable of modifying the colorassociated with dyes or colored compounds. The present inventionencompasses derivatives of natural isolates of Stachybotrys, includingprogeny and mutants, as long as the derivative is able to produce aphenol oxidizing enzyme capable of modifying the color associated withdye or color compounds.

As used herein in referring to phenol oxidizing enzymes, the term“obtainable from” means phenol oxidizing enzymes that originate from orare naturally-produced by the particular microbial strain mentioned. Toexemplify, phenol oxidizing enzymes obtainable from Stachybotrys referto those phenol oxidizing enzymes which are naturally-produced byStachybotrys. The present invention encompasses phenol oxidizing enzymesidentical to those produced by Stachybotrys species but which areproduced through the use of genetic engineering techniques by organisms,such as bacteria, fungus or yeast, transformed with a gene encoding saidphenol oxidizing enzyme or produced by organisms which are identical tothose from Stachybotrys, or equivalent to those from Stachybotrys, suchas progeny or mutants.

The present invention encompasses phenol oxidizing enzymes encoded by apolynucleotide capable of hybridizing to the polynucleotide having thesequence as shown in SEQ ID NO:1 or SEQ ID NO:3 under conditions of highstringency. The present invention encompasses polynucleotides encodingphenol oxidizing enzymes which comprises at least 65% identity, at least70%, at least 75% identity, at least 80% identity, at least 85%identity, at least 90% identity or at least 95% identity to thepolynucleotide having the sequence as disclosed in SEQ ID NO:1. Identityat the nucleic acid level is measured by the GAP program of the GCGSoftware (University Research Park, Madison, Wis.) with the followingparameters: Gap Weight=50; Length Weight=4; Gap Creation Penalty=50; andGap Extension Penalty=3. The present invention also encompasses mutants,variants and derivatives, including portions, of the phenol oxidizingenzymes of the present invention as long as the mutant, variant orderivative phenol oxidizing enzyme is able to retain at least onecharacteristic activity of the naturally occurring phenol oxidizingenzyme.

As used herein, the term ‘colored compound’ refers to a substance thatadds color to textiles or to substances which result in the visualappearance of stains. As defined in Dictionary of Fiber and TextileTechnology (Hoechst Celanese Corporation (1990) PO Box 32414, CharlotteN.C. 28232), a dye is a colored compound that is incorporated into thefiber by chemical reaction, absorption, or dispersion. Examples of dyesinclude direct Blue dyes, acid Blue dyes, direct red dyes, reactive Blueand reactive Black dyes. A catalogue of commonly used textile dyes isfound in Colour Index, 3^(rd) ed. Vol. 1-8. Examples of substances whichresult in the visual appearance of stains are polyphenols, carotenoids,anthocyanins, tannins, Maillard reaction products, etc.

As used herein the phrase “modify the color associated with a dye orcolored compound” or “modification of the colored compound” means thatthe dye or compound is changed through oxidation, either directly orindirectly, such that either the color appears modified, i.e., the colorvisually appears to be decreased, lessened, decolored, bleached orremoved, or the color is not affected but the compound is modified suchthat dye redeposition is inhibited. The present invention encompassesthe modification of the color by any means including, for example, thecomplete removal of the colored compound from stain on a fabric by anymeans as well as a reduction of the color intensity or a change in thecolor of the compound.

As used herein, the term “mutants and variants”, when referring tophenol oxidizing enzymes, refers to phenol oxidizing enzymes obtained byalteration of the naturally occurring amino acid sequence and/orstructure thereof, such as by alteration of the DNA nucleotide sequenceof the structural gene and/or by direct substitution and/or alterationof the amino acid sequence and/or structure of the phenol oxidizingenzyme. The term phenol oxidizing enzyme “derivative” as used hereinrefers to a portion or fragment of the full-length naturally occurringor variant phenol oxidizing enzyme amino acid sequence that retains atleast one activity of the naturally occurring phenol oxidizing enzyme.As used herein, the term “mutants and variants”, when referring tomicrobial strains, refers to cells that are changed from a naturalisolate in some form, for example, having altered DNA nucleotidesequence of, for example, the structural gene coding for the phenoloxidizing enzyme; alterations to a natural isolate in order to enhancephenol oxidizing enzyme production; or other changes that effect phenoloxidizing enzyme expression.

The term “enhancer” or “mediator” refers to any compound that is able tomodify the color associated with a dye or colored compound inassociation with a phenol oxidizing enzyme or a compound which increasesthe oxidative activity of the phenol oxidizing enzyme. The enhancingagent is typically an organic compound.

Phenol Oxidizing Enzymes

The phenol oxidizing enzymes of the present invention function bycatalyzing redox reactions, i.e., the transfer of electrons from anelectron donor (usually a phenolic compound) to molecular oxygen orhydrogen peroxide (which acts as an electron acceptor) which is reducedto water. Examples of such enzymes are laccases (EC 1.10.3.2), bilirubinoxidases (EC 1.3.3.5), phenol oxidases (EC 1.14.18.1), catechol oxidases(EC 1.10.3.1).

Nucleic Acid Encoding Phenol Oxidizing Enzymes

The present invention encompasses polynucleotides which encode phenoloxidizing enzymes obtainable from Stachybotrys species whichpolynucleotides comprise between at least 65% and 100% identity, that isat least 65% identity, at least 70% identity, at least 75% identity, atleast 80% identity, at least 85% identity, at least 90% identity and atleast 95% identity to the polynucleotide sequence disclosed in SEQ IDNO:3 as long as the enzyme encoded by the polynucleotide is capable ofmodifying the color associated with dyes or colored compounds. In oneembodiment, the phenol oxidizing enzyme has the polynucleotide sequenceas shown in SEQ ID NO:3 or SEQ ID NO:1 or has the polynucleotidesequence as contained in Stachybotrys chartarum having MUCL accessionnumber 38898. As will be understood by the skilled artisan, due to thedegeneracy of the genetic code, a variety of polynucleotides can encodethe phenol oxidizing enzyme disclosed in SEQ ID NO: 2. The presentinvention encompasses all such polynucleotides.

The nucleic acid encoding a phenol oxidizing enzyme may be obtained bystandard procedures known in the art from, for example, cloned DNA(e.g., a DNA “library”), by chemical synthesis, by cDNA cloning, by PCR,or by the cloning of genomic DNA, or fragments thereof, purified from adesired cell, such as a Stachybotrys species (See, for example, Sambrooket al., 1989, Molecular Cloning, A Laboratory Manual, 2d Ed., ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Glover, D. M.(ed.), 1985, DNA Cloning: A Practical Approach, MRL Press, Ltd., Oxford,U.K. Vol. I, II) Nucleic acid sequences derived from genomic DNA maycontain regulatory regions in addition to coding regions. Whatever thesource, the isolated nucleic acid encoding a phenol oxidizing enzyme ofthe present invention should be molecularly cloned into a suitablevector for propagation of the gene.

In the molecular cloning of the gene from genomic DNA, DNA fragments aregenerated, some of which will encode the desired gene. The DNA may becleaved at specific sites using various restriction enzymes.Alternatively, one may use DNAse in the presence of manganese tofragment the DNA, or the DNA can be physically sheared, as for example,by sonication. The linear DNA fragments can then be separated accordingto size by standard techniques, including but not limited to, agaroseand polyacrylamide gel electrophoresis, and column chromatography.

Once nucleic acid fragments are generated, identification of thespecific DNA fragment encoding a phenol oxidizing enzyme may beaccomplished in a number of ways. For example, a phenol oxidizing enzymeencoding gene of the present invention or its specific RNA, or afragment thereof, such as a probe or primer, may be isolated and labeledand then used in hybridization assays to detect a generated gene.(Benton, W. and Davis, R., 1977, Science 196:180; Grunstein, M. AndHogness, D., 1975, Proc. Natl. Acad. Sci. USA 72:3961). Those DNAfragments sharing substantial sequence similarity to the probe willhybridize under high stringency conditions.

The present invention encompasses phenol oxidizing enzymes obtainablefrom Stachybotrys species which are identified through nucleic acidhybridization techniques using SEQ ID NO:1 or SEQ ID NO:3 as a probe orprimer and screening nucleic acid of either genomic or cDNA origin.Nucleic acid encoding phenol oxidizing enzymes obtainable fromStachybotrys species and having at least 65% identity to SEQ ID NO:1 orSEQ ID NO:3 can be detected by DNA—DNA or DNA-RNA hybridization oramplification using probes, portions or fragments of SEQ ID NO:1 or SEQID NO:3. Accordingly, the present invention provides a method for thedetection of nucleic acid encoding a phenol oxidizing enzyme encompassedby the present invention which comprises hybridizing part or all of anucleic acid sequence of SEQ ID NO:1 or SEQ ID NO:3 with Stachybotrysnucleic acid of either genomic or cDNA origin.

Accordingly, included within the scope of the present invention arepolynucleotide sequences that are capable of hybridizing to thenucleotide sequence disclosed in SEQ ID NO:3 under conditions of highstringency. Hybridization conditions are based on the meltingtemperature (Tm) of the nucleic acid binding complex, as taught inBerger and Kimmel (1987, Guide to Molecular Cloning Techniques, Methodsin Enzymology, Vol 152, Academic Press, San Diego Calif.) incorporatedherein by reference, and confer a defined “stringency” as explainedbelow.

“Maximum stringency” typically occurs at about Tm−5° C. (5° C. below theTm of the probe); “high stringency” at about 5° C. to 10° C. below Tm;“intermediate stringency” at about 10° C. to 20° C. below Tm; and “lowstringency” at about 20° C. to 25° C. below Tm. For example in thepresent invention the following are the conditions for high stringency:hybridization was done at 37° C. in buffer containing 50% formamide,5×SSPE, 0.5% SDS and 50 ug/ml of sheared Herring DNA. The washing wasperformed at 65° C. for 30 minutes in the presence of 1×SSC and 0.1% SDSonce, at 65° C. for 30 minutes in presence of 0.5× SSC and 0.1% SDS onceand at 65° C. for 30 minutes in presence of 0.1×SSC and 0.1% SDS once;the following are the conditions for intermediate stringency:hybridization was done at 37° C. in buffer containing 25% formamide, 5×SSPE, 0.5% SDS and 50 ug/ml of sheared Herring DNA. The washing wasperformed at 50° C. for 30 minutes in presence of 1× SSC and 0.1% SDSonce, at 50° C. for 30 minutes in presence of 0.5× SSC and 0.1% SDSonce; the following are the conditions for low stringency: hybridizationwas done at 37° C. in buffer containing 25% formamide, 5× SSPE, 0.5% SDSand 50 ug/ml of sheared Herring DNA. The washing was performed at 37° C.for 30 minutes in presence of 1× SSC and 0.1% SDS once, at 37° C. for 30minutes in presence of 0.5× SSC and 0.1% SDS once. A nucleic acidcapable of hybridizing to a nucleic acid probe under conditions of highstringency will have about 80% to 100% identity to the probe; a nucleicacid capable of hybridizing to a nucleic acid probe under conditions ofintermediate stringency will have about 50% to about 80% identity to theprobe.

The term “hybridization” as used herein shall include “the process bywhich a strand of nucleic acid joins with a complementary strand throughbase pairing” (Coombs J (1994) Dictionary of Biotechnology, StocktonPress, New York N.Y.).

The process of amplification as carried out in polymerase chain reaction(PCR) technologies is described in Dieffenbach C W and G S Dveksler(1995, PCR Primer, a Laboratory Manual, Cold Spring Harbor Press,Plainview N.Y.). A nucleic acid sequence of at least about 10nucleotides and as many as about 60 nucleotides from SEQ ID NO:3preferably about 12 to 30 nucleotides, and more preferably about 25nucleotides can be used as a PCR primer.

A preferred method of isolating a nucleic acid construct of theinvention from a cDNA or genomic library is by use of polymerase chainreaction (PCR) using degenerate oligonucleotide probes prepared on thebasis of the amino acid sequence of the protein having the amino acidsequence as shown in SEQ ID NO:2. For instance, the PCR may be carriedout using the techniques described in U.S. Pat. No. 4,683,202.

Expression Systems

The present invention provides host cells, expression methods andsystems for the production of phenol oxidizing enzymes in hostmicroorganisms, such as fungus, yeast and bacteria. Once nucleic acidencoding a phenol oxidizing enzyme of the present invention is obtained,recombinant host cells containing the nucleic acid may be constructedusing techniques well known in the art. Molecular biology techniques aredisclosed in Sambrook et al., Molecular Biology Cloning: A LaboratoryManual, Second Edition (1989) Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y. (1989). Nucleic acid encoding phenol oxidizingenzymes having between at least 65% and 100%, at least 65%, at least70%, at least 75%, at least 80%, at least 85%, at least 90% and at least95% identity to the nucleic acid of SEQ ID NO:1 or SEQ ID NO:3 asmeasured by the GAP program of the GCG Software (University ResearchPark, Madison, Wis.) with the following parameters: Gap Weight=50;Length Weight=4; Gap Creation Penalty=50; and Gap Extension Penalty=3 isobtained and transformed into a host cell using appropriate vectors. Avariety of vectors and transformation and expression cassettes suitablefor the cloning, transformation and expression in fungus, yeast andbacteria are known by those of skill in the art.

Typically, the vector or cassette contains sequences directingtranscription and translation of the nucleic acid, a selectable marker,and sequences allowing autonomous replication or chromosomalintegration. Suitable vectors comprise a region 5′ of the gene whichharbors transcriptional initiation controls and a region 3′ of the DNAfragment which controls transcriptional termination. These controlregions may be derived from genes homologous or heterologous to the hostas long as the control region selected is able to function in the hostcell.

Initiation control regions or promoters, which are useful to driveexpression of the phenol oxidizing enzymes in a host cell are known tothose skilled in the art. Virtually any promoter capable of drivingthese phenol oxidizing enzyme is suitable for the present invention.Nucleic acid encoding the phenol oxidizing enzyme is linked operablythrough initiation codons to selected expression control regions foreffective expression of the oxidative or reducing enzymes. Once suitablecassettes are constructed they are used to transform the host cell.

General transformation procedures are taught in Current Protocols InMolecular Biology (vol.1, edited by Ausubel et al., John Wiley & Sons,Inc. 1987, Chapter 9) and include calcium phosphate methods,transformation using PEG and electroporation. For Aspergillus andTrichoderma, PEG and Calcium mediated protoplast transformation can beused (Finkelstein, D B 1992 Transformation. In Biotechnology ofFilamentous Fungi. Technology and Products (eds by Finkelstein & Bill)113-156. Electroporation of protoplast is disclosed in Finkelestein, DB1992 Transformation. In Biotechnology of Filamentous Fungi. Technologyand Products (eds by Finkelstein & Bill) 113-156. Microprojectionbombardment on conidia is described in Fungaro et al. (1995)Transformation of Aspergillus nidulans by microprojection bombardment onintact conidia. FEMS Microbiology Letters 125 293-298. Agrobacteriummediated transformation is disclosed in Groot et al. (1998)Agrobacterium tumefaciens-mediated transformation of filamentous fungi.Nature Biotechnology 16 839-842. For transformation of Saccharomyces,lithium acetate mediated transformation and PEG and calcium mediatedprotoplast transformation as well as electroporation techniques areknown by those of skill in the art.

Host cells which contain the coding sequence for a phenol oxidizingenzyme of the present invention and express the protein may beidentified by a variety of procedures known to those of skill in theart. These procedures include, but are not limited to, DNA—DNA orDNA-RNA hybridization and protein bioassay or immunoassay techniqueswhich include membrane-based, solution-based, or chip-based technologiesfor the detection and/or quantification of the nucleic acid or protein.

As described herein, the genomic sequence (SEQ ID NO:3) encoding phenoloxidizing enzyme obtainable from Stachybotrys chartarum (MUCL 38898) wasisolated and expressed in Aspergillus niger var. awamori and Trichodermareesei.

Phenol Oxidizing Enzyme Activities

The phenol oxidizing enzymes of the present invention are capable ofusing a wide variety of different phenolic compounds as electron donors,while being very specific for molecular oxygen or hydrogen peroxide asthe electron acceptor.

Depending upon the specific substrate and reaction conditions, e.g.,temperature, presence or absence of enhancers, etc., each phenoloxidizing enzyme oxidation reaction will have an optimum pH.

Applications Of Polyphenol Oxidizing Enzymes

As described infra, the Stachybotrys phenol oxidizing enzymes of thepresent invention are capable of oxidizing a wide variety of dyes orcolored compounds having different chemical structures, using oxygen orhydrogen peroxide as the electron acceptor. Accordingly phenol oxidizingenzymes of the present invention are used in applications where it isdesirable to modify the color associated with dyes or colored compounds,such as in cleaning, for removing the food stains on fabric; and fortextiles; and paper and pulp applications. A mediator or enhancer may beneeded to obtain desirable effects.

Colored Compounds

In the present invention, a variety of colored compounds could betargets for oxidation by phenol oxidizing enzymes of the presentinvention. For example, in detergent applications, colored substanceswhich may occur as stains on fabrics can be a target. Several types orclasses of colored substances may appear as stains, such as porphyrinderived structures, such as heme in blood stain or chlorophyll inplants; tannins and polyphenols (see P. Rib{acute over (e)}reau-Gayon,Plant Phenolics, Ed. Oliver & Boyd, Edinburgh, 1972, pp.169-198) whichoccur in tea stains, wine stains, banana stains, peach stains;carotenoids, the coloured substances which occur in tomato (lycopene,red), mango (carotene, orange-yellow) (G. E. Bartley et al., The PlantCell (1995), Vol 7, 1027-1038); anthocyanins, the highly coloredmolecules which occur in many fruits and flowers (P. Rib{acute over(e)}reau-Gayon, Plant Phenolics, Ed. Oliver & Boyd, Edinburgh, 1972,135-169); and Maillard reaction products, the yellow/brown coloredsubstances which appear upon heating of mixtures of carbohydratemolecules in the presence of protein/peptide structures, such as foundin cooking oil.

Enhancers

A phenol oxidizing enzyme of the present invention can act to modify thecolor associated with dyes or colored compounds in the presence orabsence of enhancers depending upon the characteristics of the compound.If a compound is able to act as a direct substrate for the phenoloxidizing enzyme, the phenol oxidizing enzyme will modify the colorassociated with a dye or colored compound in the absence of an enhancer,although an enhancer may still be preferred for optimum phenol oxidizingenzyme activity. For other colored compounds unable to act as a directsubstrate for the phenol oxidizing enzyme or not directly accessible tothe phenol oxidizing enzyme, an enhancer is required for optimum phenoloxidizing enzyme activity and modification of the color.

Enhancers are described in for example WO 95/01426 published Jan. 12,1995; WO 96/06930, published Mar. 7, 1996; and WO 97/11217 publishedMar. 27, 1997. Enhancers include but are not limited tophenothiazine-10-propionic acid (PTP), 10-methylphenothiazine (MPT),phenoxazine-10-propionic acid (PPO), 10-methylphenoxazine (MPO),10-ethylphenothiazine-4-carboxylic acid (EPC) acetosyringone,syringaldehyde, methylsyringate, 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonate (ABTS).

Cultures

The present invention encompasses Stachybottys strains and naturalisolates, and derivatives of such strains and isolates, such as strainsof the species Stachybotrys parvispora, including, in particular,Stachybotrys parvispora var. hughes MUCL 38996; strains of the speciesStachybotrys chartarum including, in particular, Stachybotrys chartarumMUCL 38898; S. parvispora MUCL 9485; S. chartarum MUCL 30782; S.kampalensis MUCL 39090; S. theobromae MUCL 39293; and strains of thespecies S. bisbyi, S. cylindrospora, S. dichroa, S. oenanthes and S.nilagerica which produce phenol oxidizing enzymes of the presentinvention.

The present invention provides substantially biologically-pure culturesof novel strains of the genus Stachybotrys, and, in particularsubstantially biologically-pure cultures of the strains Stachybotrysparvispora MUCL 38996 and Stachybotrys chartarum MUCL 38898 from whichphenol oxidizing enzymes can be purified.

Purification

The phenol oxidizing enzymes of the present invention may be produced bycultivation of phenol oxidizing enzyme-producing Stachybotrys strains(such as S. parvispora MUCL 38996, S. chartarum MUCL 38898) underaerobic conditions in nutrient medium containing assimiable carbon andnitrogen together with other essential nutrient(s). The medium can becomposed in accordance with principles well-known in the art.

During cultivation, the phenol oxidizing enzyme-producing strainssecrete phenol oxidizing enzyme extracellularly. This permits theisolation and purification (recovery) of the phenol oxidizing enzyme tobe achieved by, for example, separation of cell mass from a culturebroth (e.g. by filtration or centrifugation). The resulting cell-freeculture broth can be used as such or, if desired, may first beconcentrated (e.g. by evaporation or ultrafiltration). If desired, thephenol oxidizing enzyme can then be separated from the cell-free brothand purified to the desired degree by conventional methods, e.g. bycolumn chromatography.

The phenol oxidizing enzymes of the present invention may be isolatedand purified from the culture broth into which they are extracellularlysecreted by concentration of the supernatant of the host culture,followed by ammonium sulfate fractionation and gel permeationchromatography.

The phenol oxidizing enzymes of the present invention may be formulatedand utilized according to their intended application. In this respect,if being used in a detergent composition, the phenol oxidizing enzymemay be formulated, directly from the fermentation broth, as a coatedsolid using the procedure described in U.S. Pat. No. 4,689,297.Furthermore, if desired, the phenol oxidizing enzyme may be formulatedin a liquid form with a suitable carrier. The phenol oxidizing enzymemay also be immobilized, if desired.

The present invention also encompasses expression vectors andrecombinant host cells comprising a Stachybotrys phenol oxidizing enzymeof the present invention and the subsequent purification of the phenoloxidizing enzyme from the recombinant host cell.

Enzyme Compositions

A phenol oxidizing enzyme of the present invention may be used toproduce, for example, enzymatic compositions for use in detergent orcleaning compositions; in textiles, that is in the treatment,processing, finishing, polishing, or production of fibers; in theproduction of paper and pulp; and in starch processing applications.Enzymatic compositions may also comprise additional components, such asfor example, for formulation or as performance enhancers

For example, detergent composition may comprise, in addition to thephenol oxidizing enzyme, conventional detergent ingredients such assurfactants, builders and further enzymes such as, for example,proteases, amylases, lipases, cutinases, cellulases or peroxidases.Other ingredients include enhancers, stabilizing agents, bactericides,optical brighteners and perfumes. The enzymatic compositions may takeany suitable physical form, such as a powder, an aqueous or non aqueousliquid, a paste or a gel.

Having thus described the phenol oxidizing enzymes of the presentinvention, the following examples are now presented for the purposes ofillustration and are neither meant to be, nor should they be, read asbeing restrictive. Dilutions, quantities, etc. which are expressedherein in terms of percentages are, unless otherwise specified,percentages given in terms of percent weight per volume (w/v). As usedherein, dilutions, quantities, etc., which are expressed in terms of %(v/v), refer to percentage in terms of volume per volume. Temperaturesreferred to herein are given in degrees centigrade (C). The manner andmethod of carrying out the present invention may be more fullyunderstood by those of skill in the art by reference to the followingexamples, which examples are not intended in any manner to limit thescope of the present invention or of the claims directed thereto. Allreferences and patent publications referred to herein are herebyincorporated by reference.

EXAMPLE 1

Purification

This example illustrates the purification of the Stachybotrys chartarumphenol oxidizing enzyme having the amino acid sequence as shown in FIG.2.

Stachybotrys chartarum was grown on PDA plates (Difco) for about 5-10days. A portion of the plate culture (about ¾×¾ inch) was used toinoculate 100 ml of PDB (potato dextrose broth) in 500-ml shake flask.The flask was incubated at 26-28 degrees C., 150 rpm, for 3-5 days untilgood growth was obtained.

The broth culture was then inoculated into 1 L of PDB in a 2.8-L shakeflask. The flask was incubated at 26-28 degrees C., 150 rpm, for 2-4days until good growth was obtained.

A 10-L fermentor containing a production medium was prepared (containingin grams/liter the following components: glucose 15; lecithin 1.51;t-aconitic acid 1.73; KH₂PO₄ 3; MgSO₄.7H₂O 0.8; CaCl₂.2H₂O 0.1; ammoniumtartrate 1.2; soy peptone 5; Staley 7359; benzyl alcohol 1; tween 20 1;nitrilotriacetic acid 0.15; MnSO₄.7H₂O 0.05; NaCl 0.1; FeSO₄.7H₂O 0.01;CoSO₄ 0.01; CaCl₂.2H₂O 0.01; ZnSO₄.7H₂O 0.01; CuSO₄ 0.001;ALK(SO₄)2.12H₂O 0.001; H₃BO₃ 0.001; NaMoO₄.2H₂O 0.001). The fermentorwas then inoculated with the 1-L broth culture, and fermentation wasconducted at 28 degrees C. for 60 hours, under a constant air flow of5.0 liters/minute and a constant agitation of 120 RPM. The pH wasmaintained at 6.0.

The cells from one liter of broth were removed from the fermentationbroth by centrifugation and the supernatant was further clarified byfiltering through a DE filter. The low molecular weight salts wereremoved by diafiltration against 4 volumes of a buffer containing 20 mMMOPS adjusted to pH 7.0 using an Amicon YM10 membrane.

An ion exchange column containing 25 mls of Poros HQ-20 resin was usedto purify the enzyme. The column was first equilibrated with 5 columnvolumes (125 mls) of 20 mM MOPS pH 7.0. Five mls of sample containing5-10 mgs of total protein was loaded onto the column. The column wasthen washed with 3 column volumes of the MOPS buffer, then eluted with agradient of 0-0.5M ammonium sulfate in a volume of 250 mls. The flowrate was 10 mls/min. Fractions were collected in 5 mls volumes. Eachfraction was assayed for phenol oxidase activity using the ABTS method.The fractions that contained ABTS activity were subjected toelectrophoresis on SDS PAGE. The bands on the gel that corresponded tothe ABTS activity were cut out and the amino acid sequence wasdetermined.

The data shown below is from another purification run and shows thepresence of Stachybotrys oxidase B band on an SDS PAGE. In thispurification, crude material from the fermentation was purified on anion exchange column using HQ20. The fractions were subjected to aninitial non-denaturing (native) gel electrophoresis on a 4-20%Tris-Glycine gel. Samples were diluted with tracking dye and the runningbuffer was Laemmli buffer. This initial gel to look at purity was doneon all fractions of the elution peak of interest and the resulting gelwas silver stained. The second gel to confirm the active protein wasdone on every other fraction of the same peak and overlaid at pH7 andpH10 with ABTS. For the ABTS overlay, 4.5 mM ABTS was prepared at pH 7and pH 10 (pH 7 with 50 mM sodium acetate and pH 10 with 50 mM sodiumborate). The gel was divided into two parts for overlay: lanes 1-5 wereoverlaid with pH 7 and lanes 6-10 were overlaid with pH 10. Bands thatwere positive for ABTS were cut out and homogenized with Laemmli bufferand tracking dye containing BME. Samples were then placed at 100° C. for5 minutes and loaded onto a Tris-glycine 4-20% gradient gel. The runningbuffer was Laemmli with 20% SDS. The gel was then silver stained.

The results of the initial denaturing gel, the ABTS overlay gel and theSDS-PAGE gels are shown in FIGS. 8, 9 and 10, respectively.

EXAMPLE 2

Amino Acid Sequence Analysis Of Stachybotrys chartarum Phenol OxidizingEnzyme

Stachybotrys chartarum phenol oxidizing enzyme prepared as disclosed inExample 1 was subjected to SDS polyacrylamide gel electrophoresis andisolated. The isolated fraction was treated with urea and iodoacetamideand digested by the enzyme endoLysC. The fragments resulting from theendoLysC digestion were separated via HPLC (reverse phase monobore C18column, CH3CN gradient) and collected in a multititer plate. Thefractions were analysed by MALDI for mass determination and sequencedvia Edman degradation. The following amino acid sequences weredetermined and are shown in amino terminus to carboxy terminusorientation: The following amino acid sequences were determined and areshown in amino terminus to carboxy terminus orientation:

N′ FVNSGENTSPNSVHLHGSFSR C′ (SEQ ID NO:5)

N′ GVEPYEAAGLKDVVWLAR C′ (SEQ ID NO:6)

EXAMPLE 3

Cloning Genomic Nucleic Acid

Two degenerated primers were designed based on the peptide sequencesprovided in Example 2. Primer 1 contains the following sequence:GTCAACAGTGGNGARAAYAC (SEQ ID NO:7) and primer 2 contains the followingsequence: GCGGCCTCATANGGCTCNAC (SEQ ID NO:8) where N represents amixture of all four nucleotides (A, T, C and G), R represents a mixtureof A and G and Y represents a mixture of T and C.

For isolation of genomic DNA encoding phenol oxidizing enzyme, DNAisolated from Stachybotrys chartarum (MUCL #38898) was used as atemplate for PCR. The DNA was diluted 100 fold with Tris-EDTA buffer toa final concentration of 88 ng/ul. Ten microliter of diluted DNA wasadded to the reaction mixture which contained 0.2 mM of each nucleotide(A, G. C and T), 1×reaction buffer, 0.542 microgram of primer 1 and 0.62microgram of primer 2 in a total of 100 microliter reaction in aneppendorf tube. After heating the mixture at 100° C. for 5 minutes, 2.5units of Taq DNA polymerase was added to the reaction mix. The PCRreaction was performed at 95° C. for 1 minute, the primer was annealedto the template at 50° C. for 1 minute and extension was done at 72° C.for 1 minute. This cycle was repeated 30 times with an additional cycleof extension at 68° C. for 7 minutes. The PCR fragment detected byagarose gel contained a fragment of about 1.3 kilobase which was thencloned into the plasmid vector pCR-II (Invitrogen). The 1.3 kb insertwas then subjected to nucleic acid sequencing. The sequence datarevealed that it was the gene encoding Stachybotrys chartarum phenoloxidase B because the deduced peptide sequence matched the peptidesequences disclosed in Example 2 sequenced via Edman degradation. ThePCR fragments containing the 5′ gene and 3′ gene were then isolatedusing the inverse PCR method with four primers deduced based on thesequence data from the 1.3 kb PCR fragment. FIG. 3 provides the fulllength genomic sequence (SEQ ID NO:3) of the Stachybotrys phenol oxidaseB gene (spoB) including the promoter and terminator sequences.

EXAMPLE 4

Comparison Of The Stachybotrys chartarum Phenol Oxidizing Enzyme B WithOther Oxidizing Enzymes

The translated protein sequence (shown on FIG. 2) (SEQ ID NO:2) was usedas query to search DNA and protein databases. It showed thatStachybotrys oxidase B shared 67% identity to the bilirubin oxidase atthe protein sequence level. FIG. 4 shows the sequence alignment of thetwo proteins using the GAP program of GCG software (University ResearchPark, Madison, Wis.) with following parameters: Gap Weight=12; LengthWeight=4; Gap Creation Penalty=8; and Gap Extension Penalty=2.

EXAMPLE 5

Expression Of Stachybotrys Oxidase B In Aspergillus nicer var. awamori

The DNA fragment containing nucleic acid encoding the Stachybotrysphenol oxidizing enzyme B flanked by two newly introduced restrictionenzyme sites (BamHI and AgeI) was isolated by PCR. This PCR fragment wasfirst cloned into the plasmid vector pCR-II and subjected to nucleicacid sequencing to verify the gene sequence (FIG. 1). This DNA fragmentwas then cloned into the Bgl II to Age I site of vector (pGAPT2) tocreate a plasmid of pGAPT2-spoB, see FIG. 5. The expression plasmid wasdesignated as pGAPT2-spoB (FIG. 5) which is capable of integrating intothe host genome. The DNA fragment containing nucleic acid encoding theStachybotrys phenol oxidizing enzyme flanked by two newly introducedrestriction enzyme sites (BamHI and AgeI) was also cloned into theplasmid vector pRAX1 which is identical to the plasmid pGAPT2 except a5259 bp HindIII fragment of Aspergillus nidulans genomic DNA fragmentAMA1 sequence (Molecular Microbiology 1996 19:565-574) was inserted. Theexpression plasmid designated as pRAX1-spoB (FIG. 6), which is capableof being maintained as a replicating plasmid, was then transformed intoAspergillus strain GCAP4 (Gene 1990, 86:153-162) by standard PEGmethods. Transformants were selected on plates without uridine. Threetransformants were grown on -uridine plates for 3 days. The spores fromtransformants were resuspended in water with 0.01% tween80. The spores(100, 1000 or 10,000) were added to the 96 well microtiter platescontaining 160 ul of PROC medium. After 5 days growth at 30° C., thesesamples were shown to have ABTS activities. One thousand spores wereadded to 50 ml PROC medium in 250 ml shake flasks and after 3 daysgrowth at 30° C., the ABST activity was 0.33 units/ml. After 4 daysgrowth at 30° C. activity, the ABTS activity was at 4.8 units/ml. About1.2 million of spores were also added to one liter PROC medium in 2.8liter shake flasks. Production of Stachybotrys phenol oxidase B proteinreached 1 unit/ml at day 3 and 4 units/ml at day 4 and activity wasdetected in the ABTS assay.

EXAMPLE 6

Expression Of Phenol Oxidizing Enzyme In Trichoderma reesei

The expression plasmid for use in transforming Trichoderma reesei wasconstructed as follows. The ends of the BamHI to AgeI fragment shown inFIG. 5 containing the gene encoding the Stachybotrys phenol oxidizingenzyme B were blunted by T4 DNA polymerase and inserted into Pmelrestriction site of the Trichoderma expression vector, pTREX, a modifiedversion of pTEX disclosed in PCT Publication No. WO 96/23928, whichpublication is herein incorporated by reference, which contains a CBHlpromoter and terminater for gene expression and a Trichoderma pyr4 geneas a selection marker for transformants. The linear DNA fragmentcontaining only the CBH1 promoter, the phenol oxidizing gene (spoB), theCBH1 terminater and selection marker pyr4 was isolated from a gel andwas used to transform a uridine auxotroph strain of Trichoderma reesei(see U.S. Pat. No. 5,472,864) which has the four major cellulase genesdeleted. Stable transformants were isolated on Trichoderma minimalplates without uridine. The transformants were grown on 50 ml of Proflomedium in shake flasks for 4 days at 28° C. to 30° C. and expression ofthe phenol oxidizing enzyme B was assayed by ABTS as described inExample 8. Proflo medium is composed of (g/l) Proflo 22.5; lactose 30.0;(NH₄)₂SO₄ 6.5 KH₂PO₄ 2.0; MgSO₄7 H₂O 0.3; CaCL₂0.2; CaCO₃ 0.72; tracemetal stock solution 1.0 ml/l and 10% Tween 80 2.0 ml/l. The trace metalstock solution used had (g/l) FeSO₄.7H₂O 5.0; MnSO₄.H₂O 1.6; ZnSO₄.7H₂O1.4; COCl_(2.)6H₂O) 2.8.

EXAMPLE 7

Purification Of Stachybotrys Phenol Oxidase B

The Stachybotrys phenol oxidase B culture broth obtained as described inExample 5 was withdrawn from the shake flask, cooled to 4° C., andcentrifuged in a Sorval centrifuge for 15 minutes at 10,500 rpm using aGSA rotor. The resulting supernatant was then removed from the pelletand concentrated 6-10 fold by ultrafiltration using a TFF holder andcartridge UF from Millipore Corporation (6 ft{circumflex over ( )}2 PTGC10K polyethersulfone Cat.#CDUF006TG). The concentrate was washed with 4volumes of Di water by diafiltration, resulting in a recovery yieldbetween 40-80%. The material was then centrifuged again to remove thesolids, and filtered through a 0.45μ filter. The enzyme containingfiltrate was then further purified using anion exchange columnchromatography. In this regard, a Q-Sepharose anion exchange column wasequilibrated with 50 mM potassium phosphate buffer, pH 6.9. Concentrate(enzyme mixture described above) was diluted 1 part to 4 parts (5 partstotal) with 20 mM Potassium Phosphate buffer, pH 6.9 and loaded on thecolumn at 120 mL/minute. The majority of contaminants were eluted with20 mM Potassium Phosphate buffer, pH 6.9, containing 300 mM NaCl.Subsequently the column was eluted with the buffer containing 500 mMNaCl at a flow rate of 120 ml/minute. Respective fractions were thenobtained. The respective fractions containing the highest phenoloxidizing enzyme activities were pooled together, concentrated anddiafiltered to milli-Q using an Amicon concentrator with a YM10membrane. Phenol oxidizing enzyme activity was then determined using thestandard assay procedure based on the oxidation of ABTS, as described inExample 8. The enzyme activity so measured was 61.4 U/ml at pH5 and 6.1U/mL at pH9.

EXAMPLE 8

ABTS Assay

The following example describes the ABTS assay used for thedetermination of phenol oxidizing activity. The ABTS assay is aspectrophotometric activity assay which uses the following reagents:assay buffer=50 mM sodium acetate, pH 5.0; 50 mM sodium phosphate, pH7.0; 50 mM sodium carbonate, pH 9.0. The ABTS (2,2′-azinobis 3ethylbenzothiazoline-6-sulphonic acid]) was a 4.5 mM solution indistilled water. 0.75 ml assay buffer and 0.1 ml ABTS substrate solutionare combined, mixed and added to a cuvette. A cuvette containingbuffer-ABTS solution was used as a blank control. 0.05 ml of enzymesample was added, rapidly mixed and placed into the cuvette containingbuffer-ABTS solution. The rate of change in absorbance at 420 nm wasmeasured, ΔOD 420/minute, for 15 seconds (or longer for samples havingactivity rates<0.1) at 30° C. Enzyme samples having a high rate ofactivity were diluted with assay buffer to a level between 0.1 and 1.

EXAMPLE 9

Bleaching Of Tomato Stains

This example illustrates the use of the Stachybotrys phenol oxidizingenzyme having the sequence as shown in FIG. 2 in modifying the colorassociated with tomato stains.

The experiments were performed in 250 ml containers, to which 15 ml ofwash solution were added (indicated in tables). The pH of the washsolution was set to pH 9. Purified phenol oxidase from Stachybotrys wasadded to the wash solution at 6 mg/l. As the enhancersphenothiazine-10-propionate (PTP) was used, dosed at 250 mM. Thefollowing formulation was used as wash solution (2 gr/liter):

Detergent Composition: LAS 24% STP 14.5% Soda ash 17.5% silicate 8.0%SCMC 0.37% Blue pigment 0.02% Moisture/salts 34.6%

The swatches were washed for 30 minutes, at 30° C. After the wash, theswatches were tumble-dried and the reflectance spectra were measuredusing a Minolta spectrometer. The color differences between the swatchbefore and after the wash data were expressed in the CIELAB L*a*b* colorspace. In this color space, L* indicates lightness and a* and b* are thechromaticity coordinates. Color differences between two swatches areexpressed as ΔE, which is calculated from the following equation:

ΔE={square root over (ΔL ²+Δα²+Δb ²)}

The results, as ΔE values, are shown in Table 1 below:

Wash without bleach system Wash with bleach system ΔE = 6.8 ΔE = 12.2 Ascan be seen from the ΔE values, the bleaching of the tomato stain isimproved in the presence of the enzyme/enhancer system.

!

8 1 1958 DNA Stachybotrys chararum 1 ggatccatca acatgatcag ccaagctatcggagccgtgg ctctgggcct tgctgtgatc 60 ggcggcagct ctgtcgatgc cagatccgttgctggtcgat cgacagacat gccttccggt 120 ctcaccaaga ggcagacgca gctgagtcctcccctggcct tgtacgaagt gcctctgccg 180 atccctcctc tgaaggcgcc caagtagtaagtacattcta taggctagca gagccaacgt 240 tgctaatcat tgcagtaccg tccccaaccccaacactgga gaggacatct tgtactacga 300 gatggagatt aggcccttct cccaccagatctaccctgat ctggagccgg ccaacatggt 360 tggatacgat ggcatgtccc caggacctaccatcatcgtt cctcgtggca ctgagagtgt 420 tgtccgcttc gtgaacagcg gagagaacacctctcccaac agcgtccact tgcacggctc 480 tttctctcga gctccctttg atggttgggctgaggacact acccagcctg gcgagtacaa 540 ggattactac taccccaaca ggcaggctgcccgcatgctt tggtaccatg accatgccat 600 gtccatcacc gccgagaacg cctacatgggtcaggctggt gtctacatga tccaggaccc 660 ggctgaggat gccctgaacc tccccagcggctacggcgag tttgatatcc ccttggttct 720 gactgccaag cgatacaacg cagacggcactctcttctcc accaatggag aggtttccag 780 cttctggggt gacgttattc aagtggtaagttgagcccat tgagatgctt cagatcctag 840 aagtatcgat gtatgaaatt gtgcatgctctaaccagtgc tatcacagaa cggtcagcct 900 tggcctatgc tcaacgtgca gccgcgcaagtaccgcttcc gcttcctcaa cgctgccgtc 960 tcacgctctt tcgctctgta tcttgctacctctgaggatt cagagaccag acttcccttc 1020 caggtcattg ccgctgacgg tggtctgcttgagggccctg ttgacactga cactctgtac 1080 atctctatgg ccgagcgctg ggaggttgttatcgacttct ccaccttcgc tggccagtcc 1140 atcgatatcc gcaaccttcc tggtgctgacggtctcggtg ttgagcctga gtttgataac 1200 actgacaagg tcatgcgatt cgtcgttgatgaagtccttg agtcgcccga cacttctgag 1260 gtgcctgcca acctccgaga tgttcctttccccgagggcg gcaactggga ccccgcaaac 1320 cccactgatg acgagacttt caccttcggccgtgctaatg gacagtggac aatcaacgga 1380 gttaccttct cggatgtcga gaaccgtctgctccgcaatg tgccccgcga cactgttgag 1440 atctggcgac ttgagaacaa ctccaacggttggactcacc ctgttcacat tcacctcgtt 1500 gacttccgag tcctttctcg ttccactgcccgtggagtcg agccttatga ggctgctggt 1560 ctcaaggatg ttgtctggct ggctcgtcgtgaggttgtct atgttgaggc ccactacgct 1620 cctttcccgt aagttctcgc cttttacctaactggttttc actcatgcta acatctacaa 1680 gtggtgtcta catgttgcac tgccacaacctgatccacga ggaccacgac atgatggctg 1740 ctttcaatgt cactgttctc ggtgactatggctacaacta caccgagttc attgacccca 1800 tggagcctct ctggaggccc cgccccttcctcctcggaga gttcgagaat ggctcgggtg 1860 acttcagcga gcttgccatc actgaccgcattcaggagat ggctagcttc aacccctacg 1920 cccaggctga tgatgatgcc gctgaggagtagaccggt 1958 2 583 PRT Stachybotrys chartarum 2 Met Ile Ser Gln Ala IleGly Ala Val Ala Leu Gly Leu Ala Val Ile 1 5 10 15 Gly Gly Ser Ser ValAsp Ala Arg Ser Val Ala Gly Arg Ser Thr Asp 20 25 30 Met Pro Ser Gly LeuThr Lys Arg Gln Thr Gln Leu Ser Pro Pro Leu 35 40 45 Ala Leu Tyr Glu ValPro Leu Pro Ile Pro Pro Leu Lys Ala Pro Asn 50 55 60 Thr Val Pro Asn ProAsn Thr Gly Glu Asp Ile Leu Tyr Tyr Glu Met 65 70 75 80 Glu Ile Arg ProPhe Ser His Gln Ile Tyr Pro Asp Leu Glu Pro Ala 85 90 95 Asn Met Val GlyTyr Asp Gly Met Ser Pro Gly Pro Thr Ile Ile Val 100 105 110 Pro Arg GlyThr Glu Ser Val Val Arg Phe Val Asn Ser Gly Glu Asn 115 120 125 Thr SerPro Asn Ser Val His Leu His Gly Ser Phe Ser Arg Ala Pro 130 135 140 PheAsp Gly Trp Ala Glu Asp Thr Thr Gln Pro Gly Glu Tyr Lys Asp 145 150 155160 Tyr Tyr Tyr Pro Asn Arg Gln Ala Ala Arg Met Leu Trp Tyr His Asp 165170 175 His Ala Met Ser Ile Thr Ala Glu Asn Ala Tyr Met Gly Gln Ala Gly180 185 190 Val Tyr Met Ile Gln Asp Pro Ala Glu Asp Ala Leu Asn Leu ProSer 195 200 205 Gly Tyr Gly Glu Phe Asp Ile Pro Leu Val Leu Thr Ala LysArg Tyr 210 215 220 Asn Ala Asp Gly Thr Leu Phe Ser Thr Asn Gly Glu ValSer Ser Phe 225 230 235 240 Trp Gly Asp Val Ile Gln Val Asn Gly Gln ProTrp Pro Met Leu Asn 245 250 255 Val Gln Pro Arg Lys Tyr Arg Phe Arg PheLeu Asn Ala Ala Val Ser 260 265 270 Arg Ser Phe Ala Leu Tyr Leu Ala ThrSer Glu Asp Ser Glu Thr Arg 275 280 285 Leu Pro Phe Gln Val Ile Ala AlaAsp Gly Gly Leu Leu Glu Gly Pro 290 295 300 Val Asp Thr Asp Thr Leu TyrIle Ser Met Ala Glu Arg Trp Glu Val 305 310 315 320 Val Ile Asp Phe SerThr Phe Ala Gly Gln Ser Ile Asp Ile Arg Asn 325 330 335 Leu Pro Gly AlaAsp Gly Leu Gly Val Glu Pro Glu Phe Asp Asn Thr 340 345 350 Asp Lys ValMet Arg Phe Val Val Asp Glu Val Leu Glu Ser Pro Asp 355 360 365 Thr SerGlu Val Pro Ala Asn Leu Arg Asp Val Pro Phe Pro Glu Gly 370 375 380 GlyAsn Trp Asp Pro Ala Asn Pro Thr Asp Asp Glu Thr Phe Thr Phe 385 390 395400 Gly Arg Ala Asn Gly Gln Trp Thr Ile Asn Gly Val Thr Phe Ser Asp 405410 415 Val Glu Asn Arg Leu Leu Arg Asn Val Pro Arg Asp Thr Val Glu Ile420 425 430 Trp Arg Leu Glu Asn Asn Ser Asn Gly Trp Thr His Pro Val HisIle 435 440 445 His Leu Val Asp Phe Arg Val Leu Ser Arg Ser Thr Ala ArgGly Val 450 455 460 Glu Pro Tyr Glu Ala Ala Gly Leu Lys Asp Val Val TrpLeu Ala Arg 465 470 475 480 Arg Glu Val Val Tyr Val Glu Ala His Tyr AlaPro Phe Pro Gly Val 485 490 495 Tyr Met Leu His Cys His Asn Leu Ile HisGlu Asp His Asp Met Met 500 505 510 Ala Ala Phe Asn Val Thr Val Leu GlyAsp Tyr Gly Tyr Asn Tyr Thr 515 520 525 Glu Phe Ile Asp Pro Met Glu ProLeu Trp Arg Pro Arg Pro Phe Leu 530 535 540 Leu Gly Glu Phe Glu Asn GlySer Gly Asp Phe Ser Glu Leu Ala Ile 545 550 555 560 Thr Asp Arg Ile GlnGlu Met Ala Ser Phe Asn Pro Tyr Ala Gln Ala 565 570 575 Asp Asp Asp AlaAla Glu Glu 580 3 2095 DNA Stachybotrys chararum 3 cagctcggtc tactactctcgcttctcttt gacaaatcaa atctaccaat cgttccttca 60 atttcaaacg atcaacatgatcagccaagc tatcggagcc gtggctctgg gccttgctgt 120 gatcggcggc agctctgtcgatgccagatc cgttgctggt cgatcgacag acatgccttc 180 cggtctcacc aagaggcagacgcagctgag tcctcccctg gccttgtacg aagtgcctct 240 gccgatccct cctctgaaggcgcccaagta gtaagtacat tctataggct agcagagcca 300 acgttgctaa tcattgcagtaccgtcccca accccaacac tggagaggac atcttgtact 360 acgagatgga gattaggcccttctcccacc agatctaccc tgatctggag ccggccaaca 420 tggttggata cgatggcatgtccccaggac ctaccatcat cgttcctcgt ggcactgaga 480 gtgttgtccg cttcgtgaacagcggagaga acacctctcc caacagcgtc cacttgcacg 540 gctctttctc tcgagctccctttgatggtt gggctgagga cactacccag cctggcgagt 600 acaaggatta ctactaccccaacaggcagg ctgcccgcat gctttggtac catgaccatg 660 ccatgtccat caccgccgagaacgcctaca tgggtcaggc tggtgtctac atgatccagg 720 acccggctga ggatgccctgaacctcccca gcggctacgg cgagtttgat atccccttgg 780 ttctgactgc caagcgatacaacgcagacg gcactctctt ctccaccaat ggagaggttt 840 ccagcttctg gggtgacgttattcaagtgg taagttgagc ccattgagat gcttcagatc 900 ctagaagtat cgatgtatgaaattgtgcat gctctaacca gtgctatcac agaacggtca 960 gccttggcct atgctcaacgtgcagccgcg caagtaccgc ttccgcttcc tcaacgctgc 1020 cgtctcacgc tctttcgctctgtatcttgc tacctctgag gattcagaga ccagacttcc 1080 cttccaggtc attgccgctgacggtggtct gcttgagggc cctgttgaca ctgacactct 1140 gtacatctct atggccgagcgctgggaggt tgttatcgac ttctccacct tcgctggcca 1200 gtccatcgat atccgcaaccttcctggtgc tgacggtctc ggtgttgagc ctgagtttga 1260 taacactgac aaggtcatgcgattcgtcgt tgatgaagtc cttgagtcgc ccgacacttc 1320 tgaggtgcct gccaacctccgagatgttcc tttccccgag ggcggcaact gggaccccgc 1380 aaaccccact gatgacgagactttcacctt cggccgtgct aatggacagt ggacaatcaa 1440 cggagttacc ttctcggatgtcgagaaccg tctgctccgc aatgtgcccc gcgacactgt 1500 tgagatctgg cgacttgagaacaactccaa cggttggact caccctgttc acattcacct 1560 cgttgacttc cgagtcctttctcgttccac tgcccgtgga gtcgagcctt atgaggctgc 1620 tggtctcaag gatgttgtctggctggctcg tcgtgaggtt gtctatgttg aggcccacta 1680 cgctcctttc ccgtaagttctcgcctttta cctaactggt tttcactcat gctaacatct 1740 acaagtggtg tctacatgttgcactgccac aacctgatcc acgaggacca cgacatgatg 1800 gctgctttca atgtcactgttctcggtgac tatggctaca actacaccga gttcattgac 1860 cccatggagc ctctctggaggccccgcccc ttcctcctcg gagagttcga gaatggctcg 1920 ggtgacttca gcgagcttgccatcactgac cgcattcagg agatggctag cttcaacccc 1980 tacgcccagg ctgatgatgatgccgctgag gagtaaatat gatgatcgtc gaatgattta 2040 tggacagcag tatatagctattttaggaaa tacttgaata agttgtggtg cttaa 2095 4 572 PRT Stachybotryscharatum 4 Met Phe Lys His Thr Leu Gly Ala Ala Ala Leu Ser Leu Leu PheAsn 1 5 10 15 Ser Asn Ala Val Gln Ala Ser Pro Val Pro Glu Thr Ser ProAla Thr 20 25 30 Gly His Leu Phe Lys Arg Val Ala Gln Ile Ser Pro Gln TyrPro Met 35 40 45 Phe Thr Val Pro Leu Pro Ile Pro Pro Val Lys Gln Pro ArgLeu Thr 50 55 60 Val Thr Asn Pro Val Asn Gly Gln Glu Ile Trp Tyr Tyr GluVal Glu 65 70 75 80 Ile Lys Pro Phe Thr His Gln Val Tyr Pro Asp Leu GlySer Ala Asp 85 90 95 Leu Val Gly Tyr Asp Gly Met Ser Pro Gly Pro Thr PheGln Val Pro 100 105 110 Arg Gly Val Glu Thr Val Val Arg Phe Ile Asn AsnAla Glu Ala Pro 115 120 125 Asn Ser Val His Leu His Gly Ser Phe Ser ArgAla Ala Phe Asp Gly 130 135 140 Trp Ala Glu Asp Ile Thr Glu Pro Gly SerPhe Lys Asp Tyr Tyr Tyr 145 150 155 160 Pro Asn Arg Gln Ser Ala Arg ThrLeu Trp Tyr His Asp His Ala Met 165 170 175 His Ile Thr Ala Glu Asn AlaTyr Arg Gly Gln Ala Gly Leu Tyr Met 180 185 190 Leu Thr Asp Pro Ala GluAsp Ala Leu Asn Leu Pro Ser Gly Tyr Gly 195 200 205 Glu Phe Asp Ile ProMet Ile Leu Thr Ser Lys Gln Tyr Thr Ala Asn 210 215 220 Gly Asn Leu ValThr Thr Asn Gly Glu Leu Asn Ser Phe Trp Gly Asp 225 230 235 240 Val IleHis Val Asn Gly Gln Pro Trp Pro Phe Lys Asn Val Glu Pro 245 250 255 ArgLys Tyr Arg Phe Arg Phe Leu Asp Ala Ala Val Ser Arg Ser Phe 260 265 270Gly Leu Tyr Phe Ala Asp Thr Asp Ala Ile Asp Thr Arg Leu Pro Phe 275 280285 Lys Val Ile Ala Ser Asp Ser Gly Leu Leu Glu His Pro Ala Asp Thr 290295 300 Ser Leu Leu Tyr Ile Ser Met Ala Glu Arg Tyr Glu Val Val Phe Asp305 310 315 320 Phe Ser Asp Tyr Ala Gly Lys Thr Ile Glu Leu Arg Asn LeuGly Gly 325 330 335 Ser Ile Gly Gly Ile Gly Thr Asp Thr Asp Tyr Asp AsnThr Asp Lys 340 345 350 Val Met Arg Phe Val Val Ala Asp Asp Thr Thr GlnPro Asp Thr Ser 355 360 365 Val Val Pro Ala Asn Leu Arg Asp Val Pro PhePro Ser Pro Thr Thr 370 375 380 Asn Thr Pro Arg Gln Phe Arg Phe Gly ArgThr Gly Pro Thr Trp Thr 385 390 395 400 Ile Asn Gly Val Ala Phe Ala AspVal Gln Asn Arg Leu Leu Ala Asn 405 410 415 Val Pro Val Gly Thr Val GluArg Trp Glu Leu Ile Asn Ala Gly Asn 420 425 430 Gly Trp Thr His Pro IleHis Ile His Leu Val Asp Phe Lys Val Ile 435 440 445 Ser Arg Thr Ser GlyAsn Asn Ala Arg Thr Val Met Pro Tyr Glu Ser 450 455 460 Gly Leu Lys AspVal Val Trp Leu Gly Arg Arg Glu Thr Val Val Val 465 470 475 480 Glu AlaHis Tyr Ala Pro Phe Pro Gly Val Tyr Met Phe His Cys His 485 490 495 AsnLeu Ile His Glu Asp His Asp Met Met Ala Ala Phe Asn Ala Thr 500 505 510Val Leu Pro Asp Tyr Gly Tyr Asn Ala Thr Val Phe Val Asp Pro Met 515 520525 Glu Glu Leu Trp Gln Ala Arg Pro Tyr Glu Leu Gly Glu Phe Gln Ala 530535 540 Gln Ser Gly Gln Phe Ser Val Gln Ala Val Thr Glu Arg Ile Gln Thr545 550 555 560 Met Ala Glu Tyr Arg Pro Tyr Ala Ala Ala Asp Glu 565 5705 21 PRT Artificial Sequence Primer 5 Phe Val Asn Ser Gly Glu Asn ThrSer Pro Asn Ser Val His Leu His 1 5 10 15 Gly Ser Phe Ser Arg 20 6 18PRT Artificial Sequence Primer 6 Gly Val Glu Pro Tyr Glu Ala Ala Gly LeuLys Asp Val Val Trp Leu 1 5 10 15 Ala Arg 7 20 DNA Stachybotryschartarum 7 gtcaacagtg gngaraayac 20 8 20 DNA Stachybotrys chartarum 8gcggcctcat anggctcnac 20

We claim:
 1. An isolated phenol oxidizing enzyme having at least 68%identity to the phenol oxidizing enzyme having the amino acid sequenceas disclosed in SEQ ID NO:2.
 2. The phenol oxidizing enzyme of claim 1wherein said enzyme is obtainable from a Stachybotrys including S.parvispora, S. chartarum, S. kampalensis, S. theobromae, S. bisbyi, S.cylindrospora, S. dichroa, S. oenanthes and S. nilagerica.
 3. The phenoloxidizing enzyme of claim 1 having the amino acid sequence as disclosedin SEQ ID NO:2.
 4. An isolated polynucleotide encoding the amino acidhaving the sequence as shown in SEQ ID NO:2.
 5. The isolatedpolynucleotide of claim 4 having at least 65% identity to the nucleicacid sequence disclosed in SEQ ID NO: 1 or SEQ ID NO:3.
 6. The isolatedpolynucleotide of claim 5 having the nucleic acid sequence as disclosedin SEQ ID NO:1.
 7. The isolated polynucleotide of claim 5 having thenucleic acid sequence as disclosed in SEQ ID NO:3.
 8. An isolatedpolynucleotide capable of hybridizing to the polynucleotide having thesequence as shown in SEQ ID NO:1 or SEQ ID NO:3 under conditions of highstringency.
 9. An expression vector comprising the polynucleotide ofclaim
 4. 10. An expression vector comprising the polynucleotide of claim5.
 11. An expression vector comprising the polynucleotide of claim 8.12. A host cell comprising the expression vector of claim 9, claim 10,or claim
 11. 13. The host cell of claim 12 that is a filamentous fungus.14. The host cell of claim 13 wherein said filamentous fungus includesAspergillus species, Trichoderma species and Mucor species.
 15. The hostcell of claim 13 that is a yeast.
 16. The host cell of claim 15 whereinsaid yeast includes Saccharomyces, Pichia, Schizosaccharomyces,Hansenula, Kluyveromyces, and Yarrowia species.
 17. The host cell ofclaim 13 wherein said host is a bacterium.
 18. The host cell of claim 17wherein said bacterium includes Bacillus and Escherichia species.
 19. Amethod for producing a phenol oxidizing enzyme in a host cell comprisingthe steps of: a) culturing a host cell comprising a polynucleotideencoding said phenol oxidizing enzyme, wherein said enzyme has at least68% identity to the amino acid sequence disclosed in SEQ ID NO:2 underconditions suitable for the production of said phenol oxidizing enzyme;and (b) optionally recovering said phenol oxidizing enzyme produced. 20.The method of claim 19 wherein said phenol oxidizing enzyme isobtainable from a Stachybotrys including S. parvispora, S. chartarum, S.kampalensis, S. theobromae, S. bisbyi, S. cylindrospora, S. dichroa, S.oenanthes and S. nilagerica.
 21. The method of claim 19 wherein saidphenol oxidizing enzyme is obtainable from S. chartarum and has theamino acid sequence as disclosed in SEQ ID NO:2.
 22. The method of claim19 wherein said polynucleotide comprises the sequence as shown in SEQ IDNO:1 or SEQ ID NO:3.
 23. The method of claim 19 wherein said host cellincludes filamentous fungus, yeast and bacteria.
 24. The method of claim23 wherein said yeast includes Saccharomyces, Pichia,Schizosaccharomyces, Hansenula, Kluyveromyces, and Yarrowia species. 25.The method of claim 23 wherein said filamentous fungus includesAspergillus species, Trichoderma species and Mucor species.
 26. Themethod of claim 25 wherein said filamentous fungus is a species ofAspergillus.
 27. The method of claim 26 wherein the filamentous fungusis Aspergillus niger var. awamori.
 28. The method of claim 23 whereinsaid filamentous fungus is a species of Trichoderma.
 29. The method ofclaim 28 wherein said Trichoderma species is Trichoderma reseei.
 30. Amethod for producing a host cell comprising a polynucleotide encoding aphenol oxidizing enzyme, comprising the steps of: (a) obtaining apolynucleotide encoding a phenol oxidizing enzyme having at least 68%identity to the amino acid sequence disclosed in SEQ ID NO:2; (b)introducing said polynucleotide into said host cell; and (c) growingsaid host cell under conditions suitable for the production of saidphenol oxidizing enzyme.
 31. The method of claim 30 wherein said hostcell includes filamentous fungus, yeast and bacteria.
 32. The method ofclaim 31 wherein said filamentous fungus includes Aspergillus species,Trichoderma species and Mucor species.
 33. The method of claim 32wherein said Aspergillus species is Aspergillus niger var. awamori. 34.The method of claim 32 wherein said Trichoderma species is Trichodermareseei.
 35. The method of claim 31 wherein said yeast is a Saccharomycesspecies.
 36. The method of claim 35 wherein said Saccharomyces speciesis Saccharomyces cerevisiae.
 37. The method of claim 30 wherein saidpolynucleotide has at least 65% identity to the nucleic acid shown inSEQ ID NO:1 or SEQ ID NO:3.
 38. The method of claim 30 wherein saidpolynucleotide has the nucleic acid sequence as shown in SEQ ID NO:1 orSEQ ID NO:3.
 39. The method of claim 30 wherein said polynucleotide isintroduced on a replicating plasmid.
 40. The method of claim 30 whereinsaid polynucleotide is integrated into the host cell genome.
 41. Anenzymatic composition comprising the phenol oxidizing enzyme of claim 1.42. The enzymatic composition of claim 41 comprising phenol oxidizingenzyme having the sequence as shown in SEQ ID NO:2.